DESCRIPTION: The main aims of this multiple-PI proposal are: to continue to define the underlying mechanisms of improved hepatocyte transduction by our next generation of recombinant adeno-associated virus (AAV) vectors; to extend these studies to novel AAV2 variants with high liver tropism, enriched from a library, and to AAV3-based vectors, which show high efficacy in human hepatocytes and non-human primate liver; and to evaluate the efficacy and host immune response in gene therapy for hemophilia B. Candidate vectors for clinical trial with high efficacy in small and large animal models will be identified, with particular emphasis on in vivo gene transfer to human/primate hepatocytes and immunobiology. The current application encompassing an interdisciplinary approach should allow us to develop superior next generation vectors for liver-directed human gene therapy, drawing heavily from expertise at the University of Florida in AAV vector biology, immunology of AAV gene transfer, and animal model studies. We will test the following hypotheses: a. Minor changes in the capsid sequence can substantially improve liver gene transfer, in part through postentry mechanisms, allowing us to design superior AAV2- and AAV3-based vectors. b. A combination of molecular evolution and capsid engineering will result in vectors with high transduction efficiency of human/primate hepatocytes and reduced MHC I antigen presentation. c. Capsid-modified AAV vectors will provide therapeutic F.IX expression at low vector doses and with reduced immunogenicity. The following three Specific Aims will be pursued: Specific Aim 1: Further optimize and compare in vivo performance of novel AAV2- and AAV3-based vectors, and define the mechanisms responsible for the robust liver gene transfer. Specific Aim 2: Identify novel capsid variants for high-efficiency in vivo gene transfer to human hepatocytes and adopt these into an advanced baculovirus production system. Specific Aim 3: Test improved vectors in animal models of hemophilia and in non-human primates, and define their immunogenicity. These investigations have a high chance of yielding superior vectors for liver gene transfer in humans.